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Purpose: Surgical treatment of unilateral limbal stem cell deficiency (LSCD) is based on limbal stem cell transplantation. Glueless simple limbal epithelial transplantation (G-SLET) technique implements several limbal micrografts harvested from the healthy eye of the same patient into the peripheral corneal tunnels without the use of fibrin glue and human amniotic membrane., Methods: A novel customized algorithm and software for a low-energy femtosecond laser (FSL) were developed and tested using 5 pairs of isolated porcine eyes. FSL-assisted G-SLET modification was assessed in 3 clinical cases of unilateral LSCD caused by chemical burns. Corneal epithelization efficacy, best-corrected visual acuity, corneal epithelial mapping, central corneal thickness, and impression cytology with immunohistochemical examination were evaluated. All patients were followed up for 12 months postoperatively., Results: The FSL set for 100% energy and the pattern of 8 nonpenetrating vertical cuts with oblique tunnel portions having variable incision depths and diameter of 8.5 mm and higher were selected for further clinical evaluation. Clinically, stable corneal epithelialization was achieved 2 to 3 weeks after intervention. At the 6-month follow-up, all patients had a healthy corneal epithelium with limbal micrografts visible inside the corneal tunnels. Best-corrected visual acuity markedly improved in 2 cases, but not in the third case with severe corneal stromal scarring. In addition, all patients noted a full-scale reduction in subjective complaints and substantial improvement in their quality of life., Conclusions: The FSL-assisted G-SLET is a new technique for autologous limbal stem cell transplantation in patients with unilateral LSCD. It allows the standardization of corneal tunnel localization and dimensions, thereby increasing the safety of the surgical procedure., Competing Interests: M. Fabian and B. Werner are employees of the Ziemer Ophthalmic Systems manufacturer of the femtosecond laser used in this study. The authors declare that they have no conflicts of interest., (Copyright © 2024 Wolters Kluwer Health, Inc. All rights reserved.)